Comparison of N-Glycopeptide to Released N-Glycan Abundances and the Influence of Glycopeptide Mass and Charge States on N-Linked Glycosylation of IgG Antibodies.

We report the comparison of mass-spectral-based abundances of tryptic glycopeptides to fluorescence abundances of released labeled glycans and the effects of mass and charge state and in-source fragmentation on glycopeptide abundances. The primary glycoforms derived from Rituximab, NISTmAb, Evolocumab, and Infliximab were high-mannose and biantennary complex galactosylated and fucosylated N-glycans. Except for Evolocumab, in-source ions derived from the loss of HexNAc or HexNAc-Hex sugars are prominent for other therapeutic IgGs. After excluding in-source fragmentation of glycopeptide ions from the results, a linear correlation was observed between fluorescently labeled N-glycan and glycopeptide abundances over a dynamic range of 500. Different charge states of human IgG-derived glycopeptides containing a wider variety of abundant attached glycans were also investigated to examine the effects of the charge state on ion abundances. These revealed a linear dependence of glycopeptide abundance on the mass of the glycan with higher charge states favoring higher-mass glycans. Findings indicate that the mass spectrometry-based bottom-up approach can provide results as accurate as those of glycan release studies while revealing the origin of each attached glycan. These site-specific relative abundances are conveniently displayed and compared using previously described glycopeptide abundance distribution spectra "GADS" representations. Mass spectrometry data are available from the MAssIVE repository (MSV000093562).

Determining Site-Specific Glycan Profiles of Recombinant SARS-CoV-2 Spike Proteins from Multiple Sources.

Glycopeptide Abundance Distribution Spectra (GADS) were recently introduced as a means of representing, storing, and comparing glycan profiles of intact glycopeptides. Here, using that representation, an extensive analysis is made of multiple commercial sources of the recombinant SARS-CoV-2 spike protein, each containing 22 N-linked glycan sites (sequons). Multiple proteases are used along with variable energy fragmentation followed by ion trap confirmation. This enables a detailed examination of the reproducibility of the method across multiple types of variability. These results show that GADS are consistent between replicates and laboratories for sufficiently abundant glycopeptides. Derived GADS enable the examination and comparison of the glycan profiles between commercial sources of the spike protein. Multiple distinct glycopeptide distributions, generated by multiple proteases, confirm these profiles. Comparisons of GADS derived from 11 sources of recombinant spike protein reveal that sources for which protein expression methods were the same produced near-identical glycan profiles, thereby demonstrating the ability of this method to measure GADS of sufficient reliability to distinguish different glycoform distributions between commercial vendors and potentially to reliably determine and compare differences in glycosylation for any glycoprotein under different conditions of production. All mass spectrometry data files have been deposited in the MassIVE repository under the identifier MSV000091776.

Mass Spectral Library Methods for Analysis of Site-Specific N-Glycosylation: Application to Human Milk Proteins.

We present a mass spectral library-based method for analyzing site-specific N-linked protein glycosylation. Its operation and utility are illustrated by applying it to both newly measured and available proteomics data of human milk glycoproteins. It generates two varieties of mass spectral libraries. One contains glycopeptide abundance distribution spectra (GADS). The other contains tandem mass spectra of the underlying glycopeptides. Both originate from identified glycopeptides in proteolytic digests of human milk and purified glycoproteins, which include tenascin, lactoferrin, and several antibodies. Analysis was also applied to digests of a NIST human milk standard reference material (SRM), leading to a GADS library of N-glycopeptides, enabling the direct comparison of glycopeptide distributions for individual proteins. Tandem spectra underlying each glycopeptide GADS peak are combined to create a second type of library that contains spectra of the underlying glycopeptide spectra. These were acquired by higher-energy (stepped) collision dissociation fragmentation followed by ion-trap fragmentation. Spectra are annotated using MS_Piano, recently reported annotation software. This data, with extensions of a widely used spectral library search and display software, provides accessible mass spectral libraries.

Representing and Comparing Site-Specific Glycan Abundance Distributions of Glycoproteins.

A method for representing and comparing distributions of N-linked glycans located at specific sites on proteins is presented. The representation takes the form of a simple mass spectrum for a given peptide sequence, with each peak corresponding to a different glycopeptide. The mass (in place of m/z) of each peak is that of the glycan mass, and its abundance corresponds to its relative abundance in the electrospray MS1 spectrum. This provides a facile means of representing all identifiable glycopeptides arising from a single protein "sequon" on a specific sequence, thereby enabling the comparison and searching of these distributions as routinely done for mass spectra. Likewise, these reference glycopeptide abundance distribution spectra (GADS) can be stored in searchable libraries. A set of such libraries created from available data is provided along with an adapted version of the widely used NIST-MS library-search software. Since GADS contain only MS1 abundances and identifications, they are equally suitable for expressing collision-induced fragmentation and electron-transfer dissociation determinations of glycopeptide identity. Comparisons of GADS for N-glycosylated sites on several proteins, especially the SARS-CoV-2 spike protein, demonstrate the potential reproducibility of GADS and their utility for comparing site-specific distributions.

Comprehensive Analysis of Tryptic Peptides Arising from Disulfide Linkages in NISTmAb and Their Use for Developing a Mass Spectral Library.

This work presents methods for identifying and then creating a mass spectral library for disulfide-linked peptides originating from the NISTmAb, a reference material of the humanized IgG1k monoclonal antibody (RM 8671). Analyses involved both partially reduced and non-reduced samples under neutral and weakly basic conditions followed by nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS). Spectra of peptides containing disulfide bonds are identified by both MS1 ion and MS2 fragment ion data in order to completely map all the disulfide linkages in the NISTmAb. This led to the detection of 383 distinct disulfide-linked peptide ions, arising from fully tryptic cleavage, missed cleavage, irregular cleavage, complex Met/Trp oxidation mixtures, and metal adducts. Fragmentation features of disulfide bonds under low-energy collision dissociation were examined. These include (1) peptide bond cleavage leaving disulfide bonds intact; (2) disulfide bond cleavage, often leading to extensive fragmentation; and (3) double cleavage products resulting from breakages of two peptide bonds or both peptide and disulfide bonds. Automated annotation of various complex MS/MS fragments enabled the identification of disulfide-linked peptides with high confidence. Peptides containing each of the nine native disulfide bonds were identified along with 86 additional disulfide linkages arising from disulfide bond shuffling. The presence of shuffled disulfides was nearly completely abrogated by refining digest conditions. A curated spectral library of 702 disulfide-linked peptide spectra was created from this analysis and is publicly available for free download. Since all IgG1 antibodies have the same constant regions, the resulting library can be used as a tool for facile identification of "hard-to-find" disulfide-bonded peptides. Moreover, we show that one may identify such peptides originating from IgG1 proteins in human serum, thereby serving as a means of monitoring the completeness of protein reduction in proteomics studies. Data are available via ProteomeXchange with identifier PXD023358.

MS_Piano: A Software Tool for Annotating Peaks in CID Tandem Mass Spectra of Peptides and N-Glycopeptides.

Annotating product ion peaks in tandem mass spectra is essential for evaluating spectral quality and validating peptide identification. This task is more complex for glycopeptides and is crucial for the confident determination of glycosylation sites in glycoproteins. MS_Piano (Mass Spectrum Peptide Annotation) software was developed for reliable annotation of peaks in collision induced dissociation (CID) tandem mass spectra of peptides or N-glycopeptides for given peptide sequences, charge states, and optional modifications. The program annotates each peak in high or low resolution spectra with possible product ion(s) and the mass difference between the measured and theoretical m/z values. Spectral quality is measured by two major parameters: the ratio between the sum of unannotated vs all peak intensities in the top 20 peaks, and the intensity of the highest unannotated peak. The product ions of peptides, glycans, and glycopeptides in spectra are labeled in different class-type colors to facilitate interpretation. MS_Piano assists validating peptide and N-glycopeptide identification from database and library searches and provides quality control and optimizes search reliability in custom developed peptide mass spectral libraries. The software is freely available in .exe and .dll formats for the Windows operating system.

Increasing the Coverage of a Mass Spectral Library of Milk Oligosaccharides Using a Hybrid-Search-Based Bootstrapping Method and Milks from a Wide Variety of Mammals.

This study significantly expands both the scope and method of identification for construction of a previously reported tandem mass spectral library of 74 human milk oligosaccharides (HMOs) derived from results of combined LC-MS/MS experiments and comprehensive structural analysis of HMOs. In the present work, a hybrid search "bootstrap" identification method was employed that substantially broadens the coverage of milk oligosaccharides and thereby increases utility use of a spectrum library-based method for the rapid tentative identification of all distinguishable glycans in milk. This involved hybrid searching of the previous library, which was itself constructed using the hybrid search of oligosaccharide spectra in the NIST 17 Tandem MS Library. The general approach appears applicable to library construction of other classes of compounds. The coverage of oligosaccharides was significantly extended using milks from a variety of mammals, including bovine, Asian buffalo, African lion, and goat. This new method led to the identification of another 145 oligosaccharides, including an additional 80 HMOs from reanalysis of human milk. The newly identified compounds were added to a freely available mass spectral reference database of 219 milk oligosaccharides. We also provide suggestions to overcome several limitations and pitfalls in the interpretation of spectra of unknown oligosaccharides.